Molecular diagnosis in Usher syndrome is hindered by significant genetic heterogeneity, the large size of some of the Usher genes, and the number of missense changes in genes such as MYO7A
. To this is added the further potential complexity of digenic inheritance which has been proposed in some cases of Usher syndrome and described in other retinal diseases.18
Although a major undertaking in terms of time and expense, we decided at the beginning of the study to sequence all the known Usher genes in all subjects, regardless of clinical subtype, in order to assess evidence for, and contribution of, digenic inheritance and the extent of polymorphic sequence variation within the genes.
With digenic inheritance in mind, demonstrating that novel missense changes are truly pathogenic, rather than neutral variants is often difficult in the absence of functional studies; this is particularly so in the case of polymorphic genes in an ethnically diverse population. We applied a stringent assignment of pathogenicity to novel missense changes. A novel missense change was described as pathogenic only if it occurred in controls with a frequency <0.236%, was identified in trans to a pathogenic/probably pathogenic mutation, and it segregated with USH in more than two families. If the variant did not fulfil all of the mentioned criteria, it was classified as UV4/UV3 (supplemental data 1).
Although a number of molecular studies of Usher cohorts have been published to date, only one smaller study has been designed in a way that would systematically detect digenic inheritance and whether or not this is a significant or recurring phenomenon.18
Bonnet et al
described 10 (out of 54) USH patients with presumably pathogenic mutations in two different USH genes. Seven of them had biallelic mutations in one gene, and carried an additional mutation in a second and, for one of them, also a third USH gene. However, none of these had definite pathogenic mutations (ie, nonsense, frame shifting or splice) in two different genes. In all cases, one of the heterozygous
mutations was a missense change which could have been a rare benign variant or possibly a disease modifier. For example, CDH23:p.R1060W, reported as presumably pathogenic in a digenic USH case,18
has previously been published as likely non-pathogenic.20
A possible case of digenic inheritance is reported in one (out of 75) USH patients who segregated CDH23:p.T1209A and PCDH15:p.T1867del variants20
; however, the p.T1209A variant was also found in 48/904 (MAF=5.3%) alleles in the 1000 Genomes Project which suggests that it is unlikely to be pathogenic (http://browser.1000genomes.org/index.html
The polymorphic variation present in Usher genes means that multiple variants are likely to be found if multiple genes are sequenced. In our study, many patients had a number of variants across multiple genes, and there are several interesting examples of two pathogenic variants in one gene and a missense variant, previously reported as a missense mutation, in a different gene. For example, CDH23:p.Arg3175His, previously published as disease-causing,43
was identified in an USH2 family (219) together with two USH2A
truncating mutations. Another variant, more recently published as a pathogenic missense change, CDH23:p.Ala366Thr,42
occurred in 1/96 CEPH chromosomes and was found in an Usher syndrome type 1 patient 146 who has two pathogenic MYO7A
mutations (p.Asp521GlufsX8 and p.Lys1255ArgfsX8). Also CDH23:p.His755Tyr was regarded as pathogenic,18
but we identified it in a consanguineous USH2 family (203) segregating a homozygous USH2A
nonsense mutation. So although the findings of others are similar to ours, their interpretation is different. We found no convincing evidence for digenic inheritance in this study; no subject had two definitely pathogenic alleles (nonsense, frameshifts or splice mutations) in different genes, which given the overall spectrum of mutations in Usher syndrome (79% of identified pathogenic/UV4/UV3 variants were truncating mutations and 21% were missense changes) one might expect to find in genuine digenic inheritance. If digenic inheritance exists, it must be an occurrence too rare to be taken into account in genetic counselling. The only example of an USH2 patient described by Ebermann et al
, who carried a single truncating mutation in GPR98
and a truncating mutation in PDZD7
explained as ‘digenic inheritance’, could also be accounted for by an unidentified second mutation in GPR98
in combination with a modifier allele in PDZD7
. Since our study was completed before mutations in PDZD7
were published as a cause of USH, this gene was not sequenced in our cohort.
We detected at least one pathogenic/likely pathogenic mutant allele in 86% of all Usher probands studied, indicating that there is no other Usher gene of major impact in the population. However, in the USH1 cohort, only a single pathogenic/UV4/UV3 variant was identified in 4/47 (8.5%) of families and in the USH2 cohort we observed a comparatively much higher number of missing alleles with only one pathogenic/UV4/UV3 variant identified in 26/121 (21.5%) of USH2 families. Undetected large genomic rearrangements, undetected pathogenic variants in the promoter and intronic regions, misdiagnosed USH syndrome, and human as well as computer software errors during sequence analysis are likely to underlie these ‘missing alleles’. Certainly gross deletions and duplications have been well documented in genes such as PCDH15
where they account for 37% of PCDH15
and 13% of USH1 cases.36
Large genomic deletions and duplications have also been reported in MYO7A, CDH23, GPR98
, and USH2A
To analyse such rearrangements reliably, other methods such as MLPA and oligonucleotide array based comparative genomic hybridisation could be used in future.49
Our future research will focus on detection of large genomic rearrangements and mutations causing splicing aberrations at the mRNA level and will aim to clarify further the molecular diagnosis in the NCUS cohort.
Although probands with a clinical classification of Usher syndrome type 1 were screened for all USH genes, the causative mutations were only found in USH1 genes. In probands clinically classified as USH2, only 1/121 patients had a nonsense mutation in MYO7A
, an USH1 gene. In another family who entered the study with a diagnosis thought unlikely to be Usher syndrome, we identified two USH1C
mutations and affected sibs were subsequently diagnosed as having sector RP and hearing loss.35
Therefore, regarding cases with atypical presentation, the mutation detection rate is low, but even these cases can harbour mutations in the known genes and produce unexpected phenotypes. Thus clinical classification, particularly that of type 1 Usher, is generally very robust, so screening all genes is unnecessary for molecular diagnosis in most cases and segregation analysis using haplotypes will be valuable for selecting candidate genes.34
Because 52.5% of pathogenic and likely pathogenic variants were novel, the use of microarray chips for molecular diagnosis in a disorder with a large number of private mutations such as USH is limited. It can, however, serve as a useful initial screen, although hybridisation techniques are being superseded by massively parallel sequencing, with the ability to generate large datasets. The existence of LSDBs for nine Usher genes (USHbases) combining international datasets is a valuable tool for molecular genetic studies of USH. The database enables integration of published and unpublished data, is regularly updated, and currently encompasses >4500 entries with 900 unique pathogenic, neutral and unclassified variants.23
We have 295 novel variants to submit to USHbases (137 are missense changes). Integration of large datasets such as this with data from all groups studying Usher syndrome, combined with haplotype and segregation analysis in families, and functional analysis of variants, will enable more reliable detection of truly pathogenic USH variants as well as the discovery of likely modifier genes.
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